Aminopropylphosphinic acids

ABSTRACT

Intermediates used in the preparation of compounds of formula I,                  
 
or a stereoisomer thereof, or a pharmaceutically acceptable form of either selected from the group consisting of a salt, a solvate and a combination thereof,
 
with the exception of i) the racemate of (3-amino-2-hydroxypropyl)phosphinic acid; and ii) (2R/S,3R)-(3-amino-2-hydroxybutyl)phosphinic acid.

This application is a continuation of U.S. patent application Ser. No.10/406,838, filed Apr. 4, 2003 now U.S. Pat. No. 6,841,698, which is acontinuation of U.S. patent application Ser. No. 09/786,219, filed Mar.1, 2001, now U.S. Pat. No. 6,576,626, which is a §371 of internationalpatent application PCT/SE00/02426, filed Dec. 4, 2000.

FIELD OF THE INVENTION

The present invention is related to novel compounds having affinity toone or more GABA_(B) receptors, as well as to their pharmaceuticallyacceptable salts, solvates and stereoisomers. The invention is alsorelated to processes for their preparation, pharmaceutical compositionscontaining said therapeutically active compounds and to the use of saidactive compounds in therapy.

BACKGROUND AND PRIOR ART

Reflux

Gastro-oesophageal reflux disease (GORD) is the most prevalent uppergastrointestinal tract disease. Current therapy has aimed at reducinggastric acid secretion, or at reducing oesophageal acid exposure byenhancing oesophageal clearance, lower oesophageal sphincter tone andgastric emptying. The major mechanism behind reflux has earlier beenconsidered to depend on a hypotonic lower oesophageal sphincter. Howeverrecent research (e.g. Holloway & Dent (1990) Gastroenterol. Clin. N.Amer. 19, 517–535) has shown that most reflux episodes occur duringtransient lower oesophageal sphincter relaxations, hereinafter referredto as TLOSR, i.e. relaxations not triggered by swallows. It has alsobeen shown that gastric acid secretion usually is normal in patientswith GORD.

Consequently, there is a need for compounds which reduce the incidenceof TLOSR and thereby prevent reflux.

Pharmaceutical compositions comprising a local anaesthetic, adapted toinhibit relaxation of the lower oesophageal sphicter are disclosed in WO87/04077 and in U.S. Pat. No. 5,036,057. Recently GABA_(B)-receptoragonists have been shown to inhibit TLOSR which is disclosed in WO98/11885.

GABA_(B) Receptor Agonists

GABA (4-aminobutanoic acid) is an endogenous neurotransmitter in thecentral and peripheral nervous systems. Receptors for GABA havetraditionally been divided into GABA_(A) and GABA_(B) receptor subtypes.GABA_(B) receptors belong to the superfamily of G-protein coupledreceptors. GABA_(B) receptor agonists are being described as being ofuse in the treatment of CNS disorders, such as muscle relaxation inspinal spasticity, cardiovascular disorders, asthma, gut motilitydisorders such as irritable bowel syndrome (IBS) and as prokinetic andanti-tussive agents. GABA_(B) receptor agonists have also been disclosedas useful in the treatment of emesis (WO 96/11680) and recently, asmentioned above, in the inhibition of TLOSR (WO 98/11885).

The most studied GABA_(B) receptor agonist is baclofen(4-amino-3-(chlorophenyl)butanoic acid) disclosed in the Swiss patentNo. CH 449, 046. Baclofen has for several years been used as anantispastic agent. EP 0356128 describes the use of the specific compound(3-aminopropyl)methylphosphinic acid, as a potent GABA_(B) receptoragonist, in therapy. EP 0181833 discloses substituted3-aminopropylphosphinic acids which are found to have very highaffinities towards GABA_(B) receptor sites. In analogy to baclofen, thecompounds can be used as for instance muscle relaxants. EP 0399949discloses derivatives of (3-aminopropyl)methylphosphinic acid which aredescribed as potent GABA_(B) receptor agonists. These compounds arestated to be useful as muscle relaxants. EP 0463969 and FR 2722192 areboth applications related to 4-aminobutanoic acid derivatives havingdifferent heterocyclic substituents at the 3-carbon of the butyl chain.Structure-activity relationships of several phosphinic acid analogueswith respect to their affinities to the GABA_(B) receptor as well astheir muscle relaxant effect are discussed in J. Med. Chem. (1995), 38,3297–3312. The conclusion in said article is that considerably strongermuscle relaxation could be achieved with the (S)-enantiomer of3-amino-2-hydroxypropylmethylphosphinic acid than with baclofen andwithout the occurrence of unwanted CNS effects.

In literature the phosphinic acids having a hydrogen atom attached tophosphorous also are named phosphonous acids. These are two names forthe same compounds and both names can be used. However, we have chosento use the name phosphinic acids for the compounds according to thepresent invention.

OUTLINE OF THE INVENTION

The present invention provides novel compounds of the formula I

wherein

-   R₁ represents hydrogen, hydroxy, lower alkyl, lower alkoxy or    halogen;-   R₂ represents hydroxy, mercapto, halogen or an oxo group;-   R₃ represents hydrogen or lower alkyl (optionally substituted with    hydroxy, mercapto, lower alkoxy, lower thioalkoxy or aryl);-   R₄ represents hydrogen, lower alkyl (optionally substituted with    aryl), or aryl;    and pharmaceutically acceptable salts, solvates and the    stereoisomers thereof,    with the exceptions of:-   i) the racemate of (3-amino-2-hydroxypropyl)phosphinic acid, and-   ii) (2R/S,3R)-(3-amino-2-hydroxybutyl)phosphinic acid.

In a preferred embodiment

-   R₁ represents hydrogen, a lower alkyl or halogen;-   R₂ represents halogen, hydroxy or an oxo group;-   R₃ represents hydrogen; and-   R₄ represents hydrogen;    with the exception of the racemate of    (3-amino-2-hydroxypropyl)phosphinic acid.

Even more preferred compounds are (3-amino-2-fluoropropyl)phosphinicacid, (2R)-(3-amino-2-fluoropropyl)phosphinic acid,(2S)-(3-amino-2-fluoropropyl)phosphinic acid,(3-amino-2-fluoro-1-methylpropyl)phosphinic acid,(3-amino-2-oxopropyl)phosphinic acid,(2S)-(3-amino-2-hydroxypropyl)phosphinic acid,),(2R)-(3-amino-2-hydroxypropyl)phosphinic acid and(3-amino-1-fluoro-2-hydroxypropyl)phosphinic acid.

Within the scope of the invention, it is to be understood that when R₂is an oxo group the bond between R₂ and the carbon is a double bond.

Within the scope of the invention, it is to be understood by “lower”radicals and compounds, for example, those having up to and including 7,especially up to and including 4, carbon atoms. Also the general termshave the following meanings:

Lower alkyl is, for example, C₁–C₄ alkyl, such as methyl, ethyl,n-propyl or n-butyl, also isopropyl, isobutyl, secondary butyl ortertiary butyl, but may also be a C₅–C₇ alkyl group such as a pentyl,hexyl or heptyl group.

Lower alkoxy is, for example, C₁–C₄ alkoxy, such as methoxy, ethoxy,n-propoxy or n-butoxy, also isopropoxy, isobutoxy, secondary butoxy ortertiary butoxy, but may also be a C₅–C₇ alkoxy group, such as apentoxy, hexoxy or heptoxy group.

Lower thioalkoxy is, for example, C₁–C₄ thioalkoxy, such as thiomethoxy,thioethoxy, n-thiopropoxy or n-thiobutoxy, also thioisopropoxy,thioisobutoxy, secondary thiobutoxy or tertiary thiobutoxy, but may alsobe a C₅–C₇ thioalkoxy group, such as a thiopentoxy, thiohexoxy orthioheptoxy group.

Halogen is halogen of an atomic number up to and including 35, such asflourine or chlorine, and less prefered bromine.

The compounds according to formula I of the invention are of amphotericnature and may be presented in the form of internal salts. They can alsoform acid addition salts and salts with bases. Such salts areparticularly pharmaceutically acceptable acid addition salts, as well aspharmaceutically acceptable salts formed with bases. Suitable acids forthe formation of such salts include, for example, mineral acids such ashydrochloric, hydrobromic, sulfuric, or phosphoric acid or organic acidssuch as sulfonic acids and carboxylic acids. Salts with bases are, forexample, alkali metal salts, e.g. sodium or potassium salts, or alkalineearth metal salts, e.g. calcium or magnesium salts, as well as ammoniumsalts, such as those with ammonia or organic amines. The salts may beprepared by conventional methods.

When one or more stereocentre is present in the molecule, the compoundsaccording to formula I can be in the form of a stereoisomeric mixture,i.e. a mixture of diastereomers and/or racemates, or in the form of thesingle stereoisomers, i.e. the single enantiomer and/or diastereomer.The compounds can also be in the form of solvates, e.g. hydrates.

All of the compounds according to the formula I can be used for theinhibition of TLOSR, and thus for the treatment of gastro-oesophagealreflux disease. The said inhibition of TLOSR also implies that the saidcompounds of formula I can be used for the treatment of regurgitation ininfants. Effective management of regurgitation in infants would be animportant way of managing failure to thrive due to excessive loss ofingested nutrient. Furthermore the novel compounds can be used for thetreatment of GORD-related or non-GORD related asthma, belching,coughing, pain, cocaine addiction, hiccups, IBS, dyspepsia, emesis andnociception.

As opposed to what is stated in prior art, (J. Med. Chem. (1995)3297–3312 and The GABA Receptors; Second Edition, Edited by S. J. Ennaand Norman Bowery, Humana Press (1997) especially p. 281–282), thecompounds according to the invention have surprisingly high metabolicstability in spite of the presence of a P—H bond. The compounds alsopossess a surprisingly high therapeutic index.

Preparation

The compounds according to formula I of the present invention may beprepared by one of the following methods.

A) A Compound of Formula II

in which R₁ and R₃ are as defined above in formula I, X is hydrogen or aprotecting group such as —CCH₃(OCH₂CH₃)₂, Z is a protecting group suchas t-butyloxycarbonyl and Y is hydrogen or a protecting group such aslower alkyl, which compound of formula II may have been synthesized by acondensation reaction according to Scheme 1 employing an appropriateN-protected amino acid ester in which R₃ is as defined above, W is aprotecting group such as lower alkyl and Z is as defined in formula II,and a suitable protected phosphinic acid derivative in which R₁ is asdefined above in formula I, X and Y are as defined in formula II, and abase such as lithium diisopropylamide,

is

-   a) optionally converted by an N-alkylation reaction in order to    introduce R₄ if R₄ is desired to be not equal to hydrogen, and    thereafter a hydrolytic reaction to obtain a compound of formula III

wherein R₁, R₃ and R₄ are as defined above in formula I, and optionallyconvert the above resulting compound III into another chemical compoundof the formula III and/or sepatate a resulting mixture of isomers intothe individual isomers and/or convert a resulting salt into the freecompound of the formula III and/or into another salt and/or convert aresulting free compound of the formula III into a salt to correspond tothe above definition, or

-   b) converted by a reductive reaction, optionally an N-alkylation    reaction if R₄ is desired to be not equal to hydrogen, and finally a    hydrolytic reaction to obtain a compound of formula IV

wherein R₁, R₃ and R₄ are as defined above in formula I, and optionallyconvert the above resulting compound IV into another chemical compoundof the formula IV and/or sepatate a resulting mixture of isomers intothe individual isomers and/or convert a resulting salt into the freecompound of the formula IV and/or into another salt and/or convert aresulting free compound of the formula IV into a salt to correspond tothe above definition, or

-   c) converted by a reductive reaction followed by a deoxohalogenation    reaction, optionally an N-alkylation reaction in order to introduce    R₄ if R₄ is desired to be not equal to hydrogen, and finally a    hydrolytic reaction to obtain a compound of formula V

wherein R₁, R₃ and R₄ are as defined above in formula I and Halo is ahalogen atom, and optionally convert the above resulting compound V intoanother chemical compound of the formula V and/or sepatate a resultingmixture of isomers into the individual isomers and/or convert aresulting salt into the free compound of the formula V and/or intoanother salt and/or convert a resulting free compound of the formula Vinto a salt to correspond to the above definition: orB) A Compound of Formula VI

in which R₁, and R₃ are as defined above in formula I, X is hydrogen ora protecting group such as —CCH₃(OCH₂CH₃)₂, T is a group that can beconverted to a —NH₂ group, and Y is hydrogen or a protecting group suchas lower alkyl, which compound of formula VI may have been synthesizedby a condensation reaction according to Scheme 2 employing an2,3-epoxypropyl derivative, such as an appropriate N-protected2,3-epoxypropylamine derivative or an epichlorohydrin derivative, inwhich R₁ and R₃ is as defined above in formula I, and a suitableprotected phosphinic acid derivative activated by O-silylation, in whichX and Y are as defined in formula VI, and a Lewis acid such as anhydrousZnCl₂,

is

-   a) converted by a reaction where the trimethylsilyl group is    replaced by a hydrogen atom, a reaction where the T group as defined    in formula VI is converted to —NHR₄ wherein R₄ is as defined above    in formula I, and finally a hydrolytic reaction to obtain a compound    of formula IV

wherein R₁, R₃ and R₄ are as defined above in formula I, and optionallyconvert the above resulting compound IV into another chemical compoundof the formula IV and/or sepatate a resulting mixture of isomers intothe individual isomers and/or convert a resulting salt into the freecompound of the formula IV and/or into another salt and/or convert aresulting free compound of the formula IV into a salt to correspond tothe above definition, or

-   b) converted by a reaction where the trimethylsilyl group is    replaced by hydrogen, an oxidative reaction, a reaction where the T    group as defined in formula VI is converted to —NHR₄ wherein R₄ is    as defined above in formula I, and finally a hydrolytic reaction to    obtain a compound of formula III

wherein R₁, R₃ and R₄ are as defined above in formula I, and optionallyconvert the above resulting compound III into another chemical compoundof the formula III and/or sepatate a resulting mixture of isomers intothe individual isomers and/or convert a resulting salt into the freecompound of the formula III and/or into another salt and/or convert aresulting free compound of the formula III into a salt to correspond tothe above definition, or

-   c) converted by a reaction where the trimethylsilyl group is    replaced by hydrogen, a deoxohalogenation reaction, a reaction where    the T group as defined in formula VI is converted to —NHR₄ wherein    R₄ is as defined above in formula I, and finally a hydrolytic    reaction to obtain a compound of formula V

wherein R₁, R₃ and R₄ are as defined above in formula I, and Halo is ahalogen atom, and optionally convert the above resulting compound V intoanother chemical compound of the formula V and/or sepatate a resultingmixture of isomers into the individual isomers and/or convert aresulting salt into the free compound of the formula V and/or intoanother salt and/or convert a resulting free compound of the formula Vinto a salt to correspond to the above definition; orC) A Compound of Formula VII

in which R₁ is as defined above in formula I, X is hydrogen or aprotecting group such as —CCH₃(OCH₂CH₃)₂, U is an electron-withdrawinggroup, such as for instance —CN or —CO₂Et which can be converted to a—CH₂NH₂ group, and Y is hydrogen or a protecting group such as loweralkyl, and Halo is a halogen atom, which compound of formula VII mayhave been synthesized by an addition reaction according to Scheme 3employing an unsaturated compound in which R₁ is as defined above informula I, U and halo are as defined in formula VII, and a suitableprotected phosphinic acid derivative activated by O-silylation, in whichX and Y are as defined in formula VII,

is converted by a reaction where the U group is being converted to —NHR₄wherein R₄ is as defined above in formula I, and a hydrolytic reactionto obtain a compound of formula VIII

wherein R₁ and R₄ are as defined above in formula I and Halo is ahalogen atom, and optionally convert the above resulting compound VIIIinto another chemical compound of the formula VIII and/or sepatate aresulting mixture of isomers into the individual isomers and/or converta resulting salt into the free compound of the formula VIII and/or intoanother salt and/or convert a resulting free compound of the formulaVIII into a salt to correspond to the above definition; orD) A Compound of Formula IX Optionally as an Individual Stereo Isomer

in which R₁, R₃ and R₄ are as defined in formula I, Z is a protectinggroup such as t-butyloxycarbonyl and Halo is a halogen atom, whichcompound of formula IX may have been synthesized by a substitutionreaction according to Scheme 4 employing an electrophilic compound inwhich R₁, R₃ and R₄ are as defined above, L is a leaving group such asiodo, Z and Halo are as defined above, and phosphinic acid activated byO-silylation,

is converted by a hydrolytic reaction to a compound of formula V

wherein R₁, R₃ and R₄ are as defined above in formula I, and optionallyconvert the above resulting compound V into another chemical compound ofthe formula V and/or sepatate a resulting mixture of isomers into theindividual isomers and/or convert a resulting salt into the freecompound of the formula V and/or into another salt and/or convert aresulting free compound of the formula V into a salt to correspond tothe above definition; orE) A Compound of Formula XI

in which R₁, R₃ and R₄ are as defined above in formula I, X is hydrogenor a protecting group such as —CCH₃(OCH₂CH₃)₂, and Y is hydrogen or aprotecting group such as lower alkyl, which compound of formula XI mayhave been synthesized by an addition reaction according to Scheme 4treating an unsaturated phosphinic acid derivative, in which R₁, R₃ andR₄ are as defined in above in formula I, with H₂S, a mercaptide ion(HS⁻) or a protected mercapto compound such as benzyl thiol in whichcase the protective group thereafter is removed

is converted by a hydrolytic reaction to obtain a compound of formulaXII,

in which R₁, R₃ and R₄ are as defined above in formula I, and optionallyconvert the above resulting compound XII into another chemical compoundof the formula XII and/or sepatate a resulting mixture of isomers intothe individual isomers and/or convert a resulting salt into the freecompound of the formula XII and/or into another salt and/or convert aresulting free compound of the formula XII into a salt to correspond tothe above definition.

DETAILED DESCRIPTION OF THE INVENTION

The invention is described more in detail by the following non-limitingexamples

EXAMPLE 1 (3-Amino-2-fluoropropyl)phosphinic acid

To an ice bath cooled solution of ethyl(3-amino-2-fluoro-3-oxopropyl)(diethoxymethyl)phosphinate in THF(tetrahydrofuran) was added 1 M BH₃-THF while under an argon atmosphere.After 10 minutes, the solution was heated to reflux for 2.5 h. Thesolution was cooled to room temperature and 6 N HCl (200 mL) was added.The THF was removed by rotoevaporation and the aqueous layer refluxedfor 2.5 h. The solution was cooled and evaporated. The residue waspurified by ion exchange column chromatography (DOWEX® 50WX-8-200, H⁺form, 3.5×4.0 cm). The ion exchange resin was pre-washed with 2:1methanol/water (400 mL). The crude product dissolved in 1:1methanol/water was loaded onto the column and washed with 1:1methanol/water (400 mL). The eluent was changed to 3:1methanol/concentrated ammonium hydroxide. Two fractions (150 mL totally)were combined and evaporated to give 645 mg (34%) of(3-amino-2-fluoropropyl)phosphinic acid as a white solid. Data: mp203–207° C.; R_(f)=0.35 (60:40:1 methanol, methylene chloride,concentrated ammonium hydroxide); ¹H NMR (300 MHz, D₂O) δ 7.11 (d, J=528Hz, 1H), 5.18 (dm, J=54 Hz, 1H), 3.28–3.45 (m, 2H), 1.65–2.23 (m, 2H);¹³C NMR (125 MHz, D₂O+Dioxane) δ 87.8 (d, J=170 Hz), 44.3 (dd, J=12.6,21.6 Hz), 35.6 (dd, J=20.2, 86.5 Hz); APIMS: m/z=142 (M+H)⁺.

EXAMPLE 2 (2S)-(3-Amino-2-hydroxypropyl)phosphinic acid

A mixture of ethyl(2S)-(3-amino-2-hydroxypropyl)(1,1-diethoxyethyl)phosphinate (1.0 g, 3.5mmol) and concentrated HCl (50 mL) was heated to reflux for 2 h. Thesolution was cooled to room temperature and evaporated. The residue wasdissolved in methanol (100 ml) and treated with propylene oxide (2 ml)at room temperature. After the mixture was stirred for 5 hours, theprecipitated solid was collected by decanting off the solvent. The solidwas dried with a stream of argon to give 220 mg (45%) of(2S)-(3-amino-2-hydroxypropyl)phosphinic acid as a white solid. Data: ¹HNMR (300 MHz, D₂O) δ 7.1 (d, J=540 Hz, 1H), 4.2 (m, 1H), 2.9–3.2 (m,2H), 1.7–2.0 (m, 2H); ³¹P NMR (121 MHz, D₂O) δ 24.2 (d, J=522 Hz);FABMS: m/z=140 (M+H)⁺; [α]_(D) at 20° C.=+8° (0.5% in 0.1M HCl).

EXAMPLE 3 (2R)-(3-Amino-2-hvdroxypropyl)phosphinic acid

A mixture of ethyl(2R)-(3-amino-2-hydroxypropyl)(1,1-diethoxyethyl)phosphinate (0.9 g, 3.2mmol) and concentrated HCl (50 mL) was heated to reflux for 2 h. Thesolution was cooled to room temperature and evaporated. The residue wasdissolved in methanol (50 ml) and treated with propylene oxide (3 ml) atroom temperature. After the mixture was stirred for 5 hours, theprecipitated solid was collected by decanting off the solvent. The solidwas dried with a stream of argon to give 260 mg (59%) of(2R)-(3-amino-2-hydroxypropyl)phosphinic acid as a white solid. Data: ¹HNMR (300 MHz, D₂O) δ 7.1 (d, J=540 Hz, 1H), 4.2 (m, 1H), 2.9–3.2 (m,2H), 1.7–2.0 (m, 2H); ³¹P NMR (121 MHz, D₂O) δ 23.9 (d, J=525 Hz);FABMS: m/z=140 (M+H)⁺; [α]_(D) at 20° C.=−8° (0.5% in 0.1M HCl).

EXAMPLE 4 (3-Amino-2-oxopropyl)phosphinic acid

A sample of ethyl[3-[N-(tert-butoxycarbonyl)amino]-2-oxopropyl](1,1-diethoxyethyl)phosphinate(8.11 g, 21.0 mmol) was dissolved in 3 N HCl (400 mL) which waspreviously deoxygenated by bubbling N₂ through the solution. The mixturewas stirred for 14 h at room temperature and then concentrated. Theresidue was coevaporated with methanol. The residue was then dissolvedin methanol (10 mL) and propylene oxide was added (10 mL). The mixturewas stirred for 6 h and the resulting precipitate isolated byfiltration. The solid was washed with cold methanol and dried undervacuum at 50° C. to give 2.1 g (73%) of (3-amino-2-oxopropyl)phosphinicacid as an off-white solid. Data: mp 126–127° C.; R_(f)=0.64 (85:15methanol, water); ¹H NMR (300 MHz, D₂O) δ 7.13 (d, J=551 Hz, 1H), 4.14(s, 2H), 3.14 (d, J=18 Hz, 2 H); ¹³C NMR (75 MHz, D₂O+Dioxane) δ 199.5,49.2, 47.3 (d, J=69 Hz); FABMS: m/z=138 (M+H)⁺.

EXAMPLE 5 (2R)-(3-Amino-2-fluoropropyl)phosphinic acid

Ammonium hypophosphite (73.8 g, 0.89 mol) was added to 3 necked 2-Lflask equipped with a mechanical stirrer, thermometer, addition funneland an argon bubbler. The flask was placed in a water bath at roomtemperature and N,O-Bis-(trimethylsilyl)acetamide (215 mL, 0.87 mol-BSA)was added at such a rate that the internal temperature was maintainedbelow 38° C. (30 minutes approx.) using ice cooling. Upon completing theaddition of BSA, the reaction mixture was heated to 45–48° C. andmaintained at this temperature for 1 h. The reaction was cooled to roomtemperature and a solution of tert-butyl(2R)-2-fluoro-3-iodopropylcarbamate (27.3 g, 0.09 mol) in methylenechloride (300 mL) was added to the reaction mixture. The reaction wasthen allowed to stir at room temperature for 18 h. The reaction mixturewas cooled to 0° C. and was cautiously quenched with methanol (275 mL)and then with water (32 mL). The reaction mixture was stirred for 30 minafter which the reaction was filtered and the solids were washed withmethanol. The filtrate was concentrated and the residue placed underhigh vacuum (0.1 mm Hg) ovennight. The crude residue was triturated withmethylene chloride, methanol, concentrated ammonium hydroxide solution(80:20:1) and was filtered. The filtrate was concentrated under reducedpressure and the trituration was repeated. The crude concentrate wastransferred to a 2-L flask, dissolved in methanol (375 mL) and placed ina water bath at room temperature. A saturated solution of hydrogenchloride gas in ethyl acetate (500 mL) was added and the mixture stirredfor 3 h. The reaction mixture was filtered and the solids were washedwith a mixture of methanol and ethyl acetate (90:10). The filtrate wasconcentrated under reduced pressure and the crude product was passedthrough a Dowex® 50WX8-200 mesh H⁺ form (500 g, 8×15 cm) column elutingwith 1:1 methanol/water until no further material was detected by TLCanalysis. The requisite crude product was then eluted with 1:3concentrated ammonium hydroxide solution/methanol. The product wasfurther purified by column chromatography eluting with chloroform,methanol, concentrated ammonium hydroxide solution (6:3:1) to afford(2R)-(3-amino-2-fluoropropyl)phosphinic acid as a white solid (3.12 g,24%).

¹H NMR (300 MHz, D₂O) δ 7.90 (s, 0.5 H), 6.15 (s, 0.5 H), 5.12–5.29 (m,0:5 H), 4.92–5.10 (m, 0.5 H), 3.12–3.42 (m, 2H), 1.74–2.26 (m, 2H).

EXAMPLE 6 (2S)-(3-Amino-2-fluoropropyl)phosphinic acid

Ammonium hypophosphite (58.1 g, 0.70 mol) was added to a 3 necked 2-Lflask equipped with a mechanical stirrer, thermometer, addition funneland an argon bubbler. N,O-Bis-(trimethylsilyl)acetamide (175.9 mnL, 0.71mol-BSA) was added at such a rate that the internal temperature wasmaintained between 35–40° C. Upon completing the addition of BSA, thereaction mixture was maintained at 35–40° C. for 45 min. Methylenechloride (150 mL) was added and the mixture was stirred at 35–40° C. foran additional 45 min. The reaction was cooled to room temperature and asolution of tert-butyl (2S)-2-fluoro-3-iodopropylcarbamate (42.5 g, 0.14mol) in methylene chloride (300 mL) was added to the reaction mixture.The reaction was then allowed to stir at room temperature overnight. Thereaction mixture was cooled to 0° C. and was cautiously quenched withmethanol (150 mL) and then with water (60 mL). The reaction wasconcentrated and the residue placed under high vacuum (0.1 mm Hg). Theresidue was adjusted to approximately pH 8 by the addition ofconcentrated ammonium hydroxide (50 mL) then methylene chloride (400 mL)and methanol (250 mL) were added. The resulting solids were filtered andthe filtrate was concentrated. The residue was triturated with methylenechloride, methanol, concentrated ammonium hydroxide solution (80:20:1;400 mL) and was filtered. The filtrate was concentrated under reducedpressure and the crude concentrate was dissolved in methanol (400 mL). Asaturated solution of hydrogen chloride gas in ethyl acetate (600 mL)was added and the mixture stirred for 3 h. The reaction mixture wasfiltered and the filtrate was concentrated under reduced pressure. Thecrude product was passed through a Dowex® 50WX8-200 mesh H⁺ form (450 g)column eluting with 1:1 methanol/water until no further material wasdetected by TLC analysis. The requisite crude product was then elutedwith 1:3 concentrated ammonium hydroxide solution/methanol. The productwas further purified by column chromatography eluting with methylenechloride, methanol, concentrated ammonium hydroxide solution (6:3:1) toafford (2S)-(3-amino-2-fluoropropyl)phosphinic acid as a white solid(3.46 g, 17%). ¹H NMR (300 MHz, D₂O) δ 7.90 (s, 0.5 H), 6.15 (s, 0.5 H),5.12–5.29 (m, 0.5 H), 4.92–5.10 (m, 0.5 H), 3.12–3.42 (m, 2H), 1.74–2.

EXAMPLE 7 (3-Amino-1-fluoro-2-hydroxypropyl)phosphinic acid

Ethyl(3-(N-(tert-butoxycarbonyl)amino)-1-fluoro-2-hydroxypropyl)(1,1-diethoxyethyl)phosphinate(180 mg, 4.5 mmol) was dissolved in methanol (2 mL), treated with 3 NHydrochloric acid (20 mL, 60.0 mmol, sparged with argon immediatelyprior to use). The mixture was stirred at room temperature for 6 hoursunder an argon atmosphere. The reaction mixture was concentrated underreduced pressure, the crude product was re-dissolved in methanol (5 mL);the residual water removed by co-evaporation under reduced pressure withmethanol. The crude product (70 mg) was purified by columnchromatography (1×10 cm column) eluting with methylene chloride,methanol, concentrated ammonium hydroxide (6:3:1). The fractionscontaining product were concentrated under reduced pressure,co-evaporated with acetonitrile (2×10 ml) then with methanol (1×10 mL)and dried ovemight under high vacuum (0.1 mm Hg). This procedureafforded (3-amino-1-fluoro-2-hydroxypropyl)phosphinic acid as a whitesolid (40 mg, 56%). ¹H NMR (300 MHz, D₂O) δ 7.93 (s, 0.5H), 6.11 (s, 0.5H), 4.60–4.20 (m, 2H), 3.42–3.08 (m, 2H).

EXAMPLE 8 (3-Amino-2-fluoro-1-methylpropyl)phosphinic acid

To an ice bath cooled solution of ethyl3-amino-2-fluoro-1-methyl-3-oxopropyl(diethoxymethyl)phosphinate (1.6 g,5.3 mmol) in THF (15 ml) was added 1 M BH₃-THF (12.3 mL, 12.3 mmol)while under an argon atmosphere. After 10 minutes, the solution washeated to reflux for 3 h. The solution was cooled to room temperatureand 6 N HCl (100 mL) was added dropwise. The THF was removed by rotovapand another portion of 6 N HCl (100 mL) was added. The mixture wasrefluxed for 3 h. The solution was cooled, evaporated, co-evaporatedwith water and then with etanol. The residue was purified ion exchangechromatography (DOWEX® 50WX-8-200, H⁺ form, 3.5×4.0 cm). The ionexchange resin was prewashed with 2:1 methanol/water. The crude productdissolved in 1:1 methanol/water was loaded onto the column and washedwith 1:1 methanol/water. The eluent was changed to 3:1methanol/concentrated ammonium hydroxide. The appropriate fractions werecombined and evaporated to give 150 mg (15%) of a diastereomeric mixtureof (3-amino-2-fluoro-1-methylpropyl)phosphinic acid as an oil. Data: ¹HNMR (400 MHz, D₂O) δ 6.2–7.8 (m, 1H), 4.8–5.2 (m, 1H), 3.2–3.5 (m, 2H),1.8–2.2 (m, 1H), 1.0–1.2 (m, 3H); MS: m/z=156 (M+H)⁺.

The following intermediates were used in the preparation of compounds ofthe invention.

Intermediates

EXAMPLE I1 Ethyl3-[(diethoxymethyl)(ethoxy)phosphoryl]-2-fluoropropanoate (Intermediateto the Compound According to Example 1)

A mixture of ethyl (diethoxymethyl)phosphinate (26.0 g, 133 mmol) and1,1,1,3,3,3-hexamethyldisilazane (28 mL, 133 mmol) was heated to refluxfor 2 h under an argon atmosphere. The mixture was cooled to roomtemperature and fluoroacrylate (10.5 g, 89.0 mmol) was added. Thereagents were heated to 60° C. for three days under an argon atmosphere.The mixture was cooled to room temperature, diluted with ethyl acetate(300 mL), washed with 1 N HCl (2×150 mL) and saturated sodium chloride(100 mL). The organic layer was dried over MgSO₄, filtered, andevaporated to give 32.0 g of a yellow oil. The residue was purified bycoloumn chromatography on a wet-packed silica gel column (6×30 cm)eluting with 97:3 methylene chloride/methanol. The appropriate fractionswere combined and evaporated to give 16.0 g (57%) of ethyl3-[(diethoxymethyl)(ethoxy)phosphoryl]-2-fluoropropanoate as a clearoil. Data: ¹H NMR (300 MHz, CDCl₃) δ 5.32 (dm, 1H), 4.67–4.77 (m, 1H),4.18–4.32 (m, 2H), 3.58–3.91 (m, 4H), 2.30–2.62 (m, 2H), 1.20–1.41 (m,9H).

EXAMPLE I2 Ethyl(3-amino-2-fluoro-3-oxopropyl)(diethoxymethyl)phosphinate (Intermediateto Compound According to Example 1)

To a solution of ethyl3-[(diethoxymethyl)(ethoxy)phosphoryl]-2-fluoropropanoate (16.0 g, 51.1mmol) in ethanol (22 mL) was added concentrated ammonium hydroxyde (14.8N, 3.5 mL, 51.1 mmol). The solution was stirred for 16 h and evaporated.The residue was purified by chromatography on a wet-packed silica gelcolumn (7×37 cm) eluting with 96.5:3.5 methylene chloride/methanol. Theappropriate fractions were combined and evaporated to give 3.43 g (27%)of ethyl (3-amino-2-fluoro-3-oxopropyl)(diethoxymethyl)phosphinate as aclear oil. Data: ¹H NMR (300 MHz, CDCl₃) δ 6.43 (s, 1H), 5.70 (s, 1H),5.21–5.49 (dm, 1H), 4.7 (dd, 1H),4.18–4.31 (m, 2H), 3.65–3.91 (m, 4H),2.21–2.81 (m, 2H), 1.30–1.40 (m, 3H), 1.20–1.28 (m, 6H).

EXAMPLE I3 Ethyl(2R)-(3-chloro-2-hydroxypropyl)(1,1-diethoxyethyl)phosphinate(Intermediate to the Compound According to Example 2)

After a mixture of ethyl (diethoxyethyl)phosphinate (15.0 g, 71 mmol)and toluene was evaporated to dryness, the residue and1,1,1,3,3,3-hexamethyldisilazane (13.2 g, 82 mmol) was heated to refluxfor 3 h under an argon atmosphere. The mixture was cooled to room istemperature and evaporated. (R)-Epichlorohydrin (6.6 g, 71 mmol) andanhydrous zinc chloride (2.5 g, 18 mmol) were added and the reagentswere heated to 60° C. over night under an argon atmosphere. The mixturewas cooled to room temperature, diluted with methylene chloride andwater. The organic layer was washed with water, dried over MgSO₄,filtered, and evaporated to give 20.7 g of a yellow oil. The residue wasdissolved in methanol (150 mL) containing 1% acetic acid and thesolution was stirred over night. The solvent was removed to give 17.7 g(82%) of ethyl(2R)-(3-chloro-2-hydroxypropyl)(1,1-diethoxyethyl)phosphinate as a clearoil. Data: ¹H NMR (500 MHz, CDCl₃) δ 4.3–4.4 (m, 1H), 4.1–4.3 (m, 2H),3.5–3.8 (m, 4H), 1.9–2.4 (m, 2H), 1.5 (dd, J=2.3, 11.4 Hz, 3H),1.32–1.37 (m, 3H), 1.18–1.24 (m, 6H).

EXAMPLE I4 Ethyl(2S)-(3-amino-2-hydroxypropyl)(1,1-diethoxyethyl)phosphinate(Intermediate to the Compound According to Example 2)

A solution of ethyl(2R)-(3-chloro-2-hydroxypropyl)(1,1-diethoxyethyl)phosphinate (5.0 g, 17mmol) in ethanol containing 9% of ammonia was stirred in an autoclave atroom temperature for 4 days and at 60° C. for one further day. Thesolution was evaporated and the residue was purified by chromatographyon a wet-packed silica gel column eluting with methylenechloride/methanol (5–8% MeOH) containing 5% triethylamine. Theappropriate fractions were combined, evaporated and diluted withmethylene chloride and water. The aqueous layer was pH adjusted by theaddition of a few mL of 10% aqueous Na₂CO₃ and repeatedly extracted withmethylene chloride. The combined organic layers were dried over Na₂SO₄and evaporated to give 1.2 g (26%) of ethyl(2S)-(3-amino-2-hydroxypropyl)(1,1-diethoxyethyl)phosphinate as a clearoil. Data: ¹H NMR (300 MHz, CDCl₃) δ 4.40–4.55 (b, 1H), 4.10–4.30 (m,2H), 3.55–3.80 (m, 4H), 3.20–3.30 (m, 1H), 3.00–3.10 (m, 1H), 2.00–2.40(m, 2H), 1.45–1.53 (dd, J=3.4, 11.7 Hz, 3H), 1.30–1.40 (m, 3H),1.15–1.25 (m, 6H).

EXAMPLE I5 Ethyl(2S)-(3-chloro-2-hydroxypropyl)(1,1-diethoxyethyl)phosphinate(Intermediate to the Compound According to Example 3)

After a mixture of ethyl (diethoxyethyl)phosphinate (15.0 g, 71 mmol)and toluene was evaporated to dryness, the residue and1,1,1,3,3,3-hexamethyldisilazane (13.2 g, 82 mmol) was heated to refluxfor 3 h under an argon atmosphere. The mixture was cooled to roomtemperature and evaporated. (S)-Epichlorohydrin (6.6 g, 71 mmol) andanhydrous zinc chloride (2.5 g, 18 mmol) were added and the reagentswere heated to 60° C. over night under an argon atmosphere. The mixturewas cooled to room temperature, diluted with methylene chloride andwater. The organic layer was washed with water, dried over MgSO₄,filtered, and evaporated to give 20.7 g of a yellow oil. The residue wasdissolved in methanol (150 mL) containing 1% acetic acid and thesolution was stirred over night. The solvent was removed to give 16.8 g(79%) of ethyl(2S)-(3-chloro-2-hydroxypropyl)(1,1-diethoxyethyl)phosphinate as a clearoil. Data: ¹H NMR (500 MHz, CDCl₃) δ 4.4 (m, 1H), 4.2–4.3 (m, 2H),3.6–3.8 (m, 4H), 1.9–2.4 (m, 2H), 1.5 (dd, J=2.3, 11.4 Hz, 3H),1.32–1.37 (m, 3H), 1.18–1.24 (m, 6H).

EXAMPLE I6 Ethyl(2R)-(3-amino-2-hydroxypropyl)(1,1-diethoxyethyl)phosphinate(Intermediate to the Compound According to Example 3)

A solution of ethyl(2S)-(3-chloro-2-hydroxypropyl)(1,1-diethoxyethyl)phosphinate (5.0 g, 17mmol) in ethanol containing 9% of ammonia was stirred in an autoclave atroom temperature for 6 days and at 55° C. for one further day. Thesolution was evaporated and the residue was purified by chromatographyon a wet-packed silica gel column eluting with methylenechloride/methanol (5–8% MeOH) containing 5% triethylamine. Theappropriate fractions were combined, evaporated and diluted withmethylene chloride and water. The aqueous layer was pH adjusted by theaddition of a few mL of 10% aqueous Na₂CO₃ and repeatedly extracted withmethylene chloride. The combined organic layers were dried over Na₂SO₄and evaporated to give 0.9 g (19%) of ethyl(2R)-(3-amino-2-hydroxypropyl)(1,1-diethoxyethyl)phosphinate as a clearoil. Data: ¹H NMR (500 MHz, CDCl₃) δ 4.1–4.3 (m, 2H), 4.05 (b, 1H),3.60–3.80 (m, 4H), 2.4–2.9 (m, 2H), 1.7–2.1 (m, 2H), 1.4–1.5 (dd,3H),1.3–1.4 (m, 3H), 1.2 (m, 6H).

EXAMPLE I7 Ethyl[3-[N-(tert-butoxycarbonyl)amino]-2-oxopropyl](1,1-diethoxyethyl)phosphinate(Intermediate to the Compound According to the Example 4)

To a solution of diisopropylamine (3.0 mL, 21 mmol) in THF (5 mL) at−10° C. was added dropwise n-BuLi (2.5 M in hexanes, 8.6 mL, 21 mmol).After 10 minutes, the reaction was cooled to −78° C. and a solutionethyl (1,1-diethoxyethyl)(methyl)phosphinate (4.80 g, 21.0 mmol) in THF(5 mL) was added dropwise. After the addition, the solution was stirredat −78° C. for 1 h. A solution of N-Boc-glycine methyl ester (810 mg,4.3 mmol) in THF (15 mL) was added dropwise. After the addition wascomplete, the reaction mixture was stirred for 45 minutes. Acetic acid(1.2 mL, 21 mmol) was added and the reaction mixture was warmed to roomtemperature. The reaction mixture was partitioned between methylenechloride and water and the layers were separated. The aqueous layer wasextracted once with methylene chloride. The combined organic extractswere dried over MgSO₄, filtered, and evaporated to give 4.89 g of anoil. The residue was purified by chromatography on 100 g of silica geleluting with ethyl acetate. The appropriate fractions were collected togive 1.2 g (74%) of ethyl[3-[N-(tert-butoxycarbonyl)amino]-2-oxopropyl)(1,1-diethoxyethyl)phosphinateas an oil. Data: ¹H NMR (300 MHz, CDCl₃) δ 5.48 (s, 1H), 4.10–4.30 (m,2H), 4.17 (d, 2H), 3.60–3.80 (m, 4H), 3.01–3.30 (m, 2H), 1.52 (d, 3H),1.43 (s, 9H), 1.32 (t, 3H), 1.19 (t, 6H).

EXAMPLE I8 (2R)-3-(Dibenzylamino)-2-fluoro-1-propanol (Intermediate tothe Compound According to the Example 5)

Lithium borohydride (5.3 g, 0.24 mol) was suspended in THF (200 mL)under a nitrogen atmosphere and cooled to −15° C. with stirring. Methyl(2R)-3-(dibenzylamino)-2-fluoropropanoate (56.6 g, 0.19 mol) wassuspended in THF (250 mL) and added dropwise to the mixture over 1 h;the internal temperature was maintained below −10° C. during theaddition. On completion of addition, the reaction mixture was allowed towarm to room temperature and stirred at this temperature for 17 h. Thereaction mixture was cooled to 0° C. and cautiously quenched with asaturated aqueous solution of ammonium chloride (300 mL). The reactionmixture was extracted with ethyl acetate (2×200 mL) and the organicphase was concentrated under reduced pressure. The crude residue wasdissolved in 2 N hydrochloric acid (200 mL, pH=2 approx.) and theaqueous phase was washed with ether (2×200 mL). The aqueous phase wasbasified (pH=10 approx.) with 80% ammonium hydroxide in brine, extractedwith ethyl acetate (3×200 mL), dried over anhydrous sodium sulfate (10g), filtered and concentrated under reduced pressure to afford(2R)-3-(dibenzylamino)-2-fluoro-1-propanol (48 g, 93%) as a yellow oil.

¹H NMR (300 MHz, CDCl₃) δ 7.15–7.38 (m, 10H), 4.65–4.78 (m, 0.5H),4.48–4.58 (m, 0.5H), 3.50–3.82 (m, 6H), 2.70–2.88 (m, 2H).

EXAMPLE I9 (2R)-3-Amino-2-fluoro-1-propanol (Intermediate to theCompound According to the Example 5)

(2R)-3-(dibenzylamino)-2-fluoro-1-propanol (29.2 g, 0.11 mol) wasdissolved in ethanol (300 mL). Ten wt. % Palladium (II) hydroxide oncarbon (5.0 g) was added and the mixture placed on a Parr® shaker andshaken under a hydrogen atmosphere (55 psi) for 6 h. When no furtherhydrogen uptake was observed, the mixture was filtered through a pad ofCelite® (20 g). A fresh batch of palladium (II) hydroxide (5 g) wasadded to the ethanol mixture and re-subjected to the hydrogenationconditions described above for 17 h. The crude reaction mixture wasfiltered through Celite® and concentrated under reduced pressure toafford (2R)-3-amino-2-fluoro-1-propanol as a pale yellow oil (9.6 g,96%).

¹H NMR (300 MHz, CD₃OD) δ 4.78–5.00 (br s, 3H), 4.49–4.62 (m, 0.5H),4.32–4.46 (m, 0.5H), 3.54–3.70 (m, 2H), 2.70–2.96 (m, 2H).

EXAMPLE I10 Tert-butyl (2R)-2-fluoro-3-hydroxypropylcarbamate(Intermediate to the Compound According to the Example 5)

(2R)-3-amino-2-fluoro-1-propanol (4.6 g, 49 mmol) was dissolved in 25%aqueous dioxane (160 mL), potassium carbonate (7.1 g, 51 mmol) was addedand the mixture cooled to 0° C. Di-tert-butyl dicarbonate (11.6 g, 53mmol) was added in two portions. The mixture was then allowed to warm toroom temperature overnight. The crude reaction mixture was concentratedto dryness, water (150 mL) was added followed by saturated aqueouspotassium hydrogen sulfate (until pH=3 approx.). The organic materialwas extracted with methylene chloride (2×150 mL), dried over sodiumsulfate, filtered and concentrated under reduced pressure to affordtert-butyl (2R)-2-fluoro-3-hydroxypropylcarbamate (9.5 g, 100%) as acolorless oil.

¹H NMR (300 MHz, CDCl₃) δ 4.82–5.04 (br s, 1H), 4.62–4.72 (m, 0.5H),4.48–4.58 (m, 0.5H), 3.62–3.72 (m, 2H), 3.32–3.62 (m, 2H), 3.20–3.44 (brs, 1H), 1.48 (s, 9H).

EXAMPLE I11 Tert-butyl (2R)-2-fluoro-3-iodopropylcarbamate (Intermediateto the Compound According to the Example 5)

Imidazole (26.6 g, 0.39 mol) was dissolved in methylene chloride (400mL) at room temperature. Iodine (102.5 g, 0.39 mol) was added and thereaction mixture was stirred for 10 min at room temperature and thencooled to 0° C. Triphenylphosphine (102.5 g, 0.39 mol) was addedportionwise over 10 min such that the internal temperature remainedbelow 10° C. A solution of tert-butyl(2R)-2-fluoro-3-hydroxypropylcarbamate (60.4 g, 0.31 mol) in methylenechloride (100 mL) was added dropwise. On completion of addition oftert-butyl (2R)-2-fluoro-3-hydroxypropylcarbamate, additional methylenechloride (200 mL) was added. The reaction mixture was allowed to warm toroom temperature and stirring was continued for 17 h. The reactionmixture was filtered through a pad of Celite® (50 g) and washed withadditional methylene chloride. The filtrate was concentrated underreduced pressure and purified by silica gel column chromatographyeluting with methylene chloride. This procedure afforded tert-butyl(2R)-2-fluoro-3-iodopropylcarbamate as a white solid (64.7 g, 68%).

¹H NMR (300 MHz, CDCl₃) δ 4.80–5.10 (br s, 1H), 4.58–4.72 (m, 0.5H),4.42–4.56 (m, 0.5 H), 3.48–3.70 (m, 1H), 3.20–3.46 (m, 3H), 1.48 (s,9H).

EXAMPLE I12 Methyl (2S)-3-(dibenzylamino)-2-fluoropropanoate(Intermediate to the Compound According to the Example 6)

Methyl (2R)-2-(dibenzylamino)-3-hydroxypropanoate (231.7 g, 0.77 mol)was dissolved in THF (850 mL) and a solution of DAST (196 g, 1.2 mol) inTHF (400 mL) was added slowly dropwise. Once the addition was complete,the reaction was stirred for an additional 1.5 h. TLC analysis indicatedconsumption of starting material. The reaction was then cooled to 0° C.and was quenched by the slow addition of water (1.5 l) followed byneutralization by the addition of solid sodium bicarbonate. Onceneutral, a 1:1 mixture of concentrated ammonium hydroxide/saturatedsodium chloride solution was added and the reaction was extracted withethyl acetate and concentrated under reduced pressure. The crude mixturewas purified by silica gel column chromatography eluting with ethylacetate, hexanes (1:4) to provide the desired compound (188.3 g, 62%) asan oil.

¹H NMR (300 MHz, CDCl₃) δ 7.18–7.38 (m, 10H), 5.12–5.17 (m, 0.5H),4.95–5.00 (m, 0.5H), 3.81–3.87 (m, 2H), 3.69 (s, 3H), 3.49–3.55 (m, 2H),2.90–3.12 (m, 2H).

EXAMPLE I13 (2S)-3-(Dibenzylamino)-2-fluoro-1-propanol (Intermediate tothe Compound According to the Example 6)

Lithium borohydride (17.7 g, 0.81 mol) was suspended in THF (400 mL)under a nitrogen atmosphere and cooled to −15° C. with stirring. Methyl(2S)-3-(dibenzylamino)-2-fluoropropanoate (188.3 g, 0.62 mol) wassuspended in THF (400 mL) and added dropwise to the mixture. Oncompletion of addition, the reaction mixture was allowed to warm to roomtemperature and stirred at this temperature for 3 h. TLC analysisindicated complete consumption of starting material. The reactionmixture was cooled to 0° C. and cautiously quenched with a saturatedaqueous solution of ammonium chloride (300 mL). Additional water (400mL) was added then the reaction mixture was extracted with ethyl acetateand the organic phase was concentrated under reduced pressure. The cruderesidue was dissolved in 2 N hydrochloric acid and the aqueous phase waswashed twice with ether. The aqueous phase was basified (pH=10 approx.)with 80% ammonium hydroxide in brine, extracted with ethyl acetate,dried over anhydrous sodium sulfate, filtered and concentrated underreduced pressure to afford (2S)-3-(dibenzylamino)-2-fluoro-1-propanol(156.6 g, 92%) as a yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 7.15–7.38 (m,10H), 4.65–4.78 (m, 0.5H), 4.48–4.58 (m, 0.5H), 3.50–3.82 (m, 6H),2.70–2.88 (m, 2H).

EXAMPLE I14 (2S)-3-Amino-2-fluoro-1-propanol (Intermediate to theCompound According to the Example 6)l

(2S)-3-(dibenzylamino)-2-fluoro-1-propanol (39.1 g, 0.14 mol) wasdissolved in ethanol (300 mL). Ten wt. % Palladium (II) hydroxide oncarbon (5.0 g) was added and the mixture placed on a Parr® shaker andshaken under a hydrogen atmosphere (55 psi) overnight. When no furtherhydrogen uptake was observed, the mixture was filtered through a pad ofCelite®. A fresh batch of palladium (II) hydroxide (5 g) was added tothe ethanol mixture and re-subjected to the hydrogenation conditionsdescribed above for 12 h. Again, when no further hydrogen uptake wasobserved, the mixture was filtered through a pad of Celite®. A freshbatch of palladium (II) hydroxide (5 g) was added to the ethanol mixtureand re-subjected to the hydrogenation conditions described above for 12h. The crude reaction mixture was filtered through Celite® andconcentrated under reduced pressure to afford(2S)-3-amino-2-fluoro-1-propanol as a pale yellow oil (13.3 g, 100%). ¹HNMR (300 MHz, CD₃OD) δ 4.78–5.00 (br s, 3H), 4.49–4.62 (m, 0.5H),4.32–4.46 (m, 0.5H), 3.54–3.70 (m, 2H), 2.70–2.96 (m, 2H).

EXAMPLE I15 Tert-butyl (2S)-2-fluoro-3-hydroxypropylcarbamate(Intermediate to the Compound According to the Example 6

(2S)-3-amino-2-fluoro-1-propanol (38.6 g, 0.41 mol) was dissolved in 25%aqueous dioxane (1.4 L), potassium carbonate (60.1 g, 0.43 mol) wasadded followed by di-tert-butyl dicarbonate (99.5 g, 0.46 mol). Themixture was stirred overnight. TLC analysis indicated completeconsumption of starting material. The crude reaction mixture wasconcentrated to dryness, water (300 mL) was added followed by saturatedaqueous potassium hydrogen sulfate (until pH=3 approx.). The organicmaterial was extracted twice with methylene chloride, dried over sodiumsulfate, filtered and concentrated under reduced pressure to affordtert-butyl (2S)-2-fluoro-3-hydroxypropylcarbamate (79.5 g, 99%) as apale yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 4.82–5.04 (br s, 1H),4.62–4.72 (m, 0.5H), 4.48–4.58 (m, 0.5H), 3.62–3.72 (m, 2H), 3.32–3.62(m, 2H), 3.20–3.44 (br s, 1H), 1.48 (s, 9H).

EXAMPLE I16 Tert-butyl (2S)-2-fluoro-3-iodopropylcarbamate (Intermediateto the Compound According to the Example 6)

Imidazole (19.8 g, 0.29 mol) was dissolved in methylene chloride (900mL) at room temperature. Iodine (73.9 g, 0.29 mol) was added and thereaction mixture was stirred for 10 min at room temperature and thencooled to 0° C. Triphenylphosphine (76.3 g, 0.29 mol) was addedportionwise over 10 min such that the internal temperature remainedbelow 10° C. A solution of tert-butyl(2S)-2-fluoro-3-hydroxypropylcarbamate (45.0 g, 0.23 mol) in methylenechloride (300 mL) was added dropwise. The reaction mixture was allowedto warm to room temperature and stirring was continued for 12 h. Thereaction mixture was filtered through a pad of Celite® and washed withadditional methylene chloride. The filtrate was concentrated underreduced pressure and purified by silica gel column chromatographyeluting with methylene chloride. This procedure afforded tert-butyl(2S)-2-fluoro-3-iodopropylcarbamate as a colorless oil (42.5 g, 62%). ¹HNMR (300 MHz, CDCl₃) δ 4.80–5.10 (br s, 1H), 4.58–4.72 (m, 0.5H),4.42–4.56 (m, 0.5 H), 3.48–3.70 (m, 1H), 3.20–3.46 (m, 3H), 1.48 (s,9H).

EXAMPLE I17 Ethyl (fluoromethyl)(1,1-diethoxyethyl)phosphinate(Intermediate to the Compound According to the Example 7)

Sodium hydride (1.4 g, 57.1 mmol) was suspended in THF (50 mL) in apressure flask under a nitrogen atmosphere and cooled to −10° C. withstirring. Ethyl (1,1-diethoxyethyl)phosphinate (10.0 g, 47.6 mmol) inTHF (20 mL) was added dropwise to the mixture over 10 minutes; theinternal temperature was maintained below 0° C. during the addition. Oncompletion of addition, the reaction mixture was allowed to stir at thistemperature for 90 minutes. The flask was cooled to −78° C. andchlorofluoromethane gas (9.7 g, 142.8 mmol) was condensed into thereaction mixture. The septum was removed and the flask was sealed with ascrew thread stopper. The flask was then allowed to warm to roomtemperature and then heated at 50° C. for 24 h. The reaction mixture wascooled to 0° C. and cautiously quenched with water (25 mL). Methylenechloride (50 mL) was added to the reaction mixture and the emulsion wasfiltered through a pad of Celite ® (20 g). The aqueous phase wasextracted with methylene chloride (2×100 mL), dried over anhydrousmagnesium sulfate, and the organic phase was concentrated under reducedpressure affording the crude product as a pale yellow oil (6.93 g). Thecrude residue was purified by silica gel column chromatography (6×25 cmcolumn) eluting with 20% acetone in hexanes. This procedure affordedethyl (fluoromethyl)(1,1-diethoxyethyl)phosphinate as a clear, colorlessoil (4.4 g, 42%). ¹H NMR (300 MHz, CDCl₃) δ 4.94–4.54 (m, 2H), 4.32–4.20(m, 2H), 3.82–3.54 (m, 4H), 1.60–1.44 (m, 3H), 1.40–1.28 (m, 3H),1.26–1.08 (m, 6H).

EXAMPLE I18 Ethyl(3-(N-(tert-butoxycarbonyl)amino)-1-fluoro-2-oxopropyl)(1,1-diethoxyethyl)phosphinate(Intermediate to the Compound According to the Example 7)

To a solution of diisopropylamine (2.5 mL, 14.5 mmol, 3.5 eq) in THF (30mL) at −10° C. was added dropwise (about 10 minutes) n-BuLi (1.4 M inhexanes, 9.0 mL, 14.5 mmol). After 10 minutes, the reaction was cooledto −78° C., and a solution of ethyl(fluoromethyl)(1,1-diethoxyethyl)phosphinate (2.0 g, 8.26 mmol, 2 eq) inTHF (10 mL) was added dropwise over 10 minutes. After the addition thereaction mixture was stirred at −78° C. for 1 h. A solution ofN-Boc-glycine methyl ester (0.8 g, 4.1 mmol) in THF (10 nL) was addeddropwise over 10 minutes such that the internal temperature wasmaintained below −70° C. After the addition was complete the reactionwas stirred at −78° C. for 1 h. The reaction was quenched with aceticacid (1 mL, 14.5 mmol) and then warmed to room temperature. Saturatedaqueous sodium chloride (75 mL) was added to the reaction and theorganic phase separated. The aqueous phase was then extracted with ethylacetate (2×75 mL). The combined, organic phase was dried over anhydroussodium sulfate and concentrated under reduced pressure affording thecrude product as a pale yellow oil (2.69 g). The crude product waspurified by column chromatography (2×35 cm column), eluting with 40%ethyl acetate in hexane. This method afforded ethyl(3-(N-(tert-butoxycarbonyl)amino)-1-fluoro-2-oxopropyl)(1,1-diethoxyethyl)phosphinateas a clear colorless oil (0.73 g, 44%). ¹H NMR (300 MHz, CDCl₃) δ5.78–5.24 (m,2H), 4.52–4.08 (m, 4H), 3.94–3.50 (m, 4H), 1.62–1.51 (m,3H), 1.50–1.32 (m, 3H), 1.42 (s, 9H), 1.30–1.12 (m, 6H).

EXAMPLE I19 Ethyl(3-(N-(tert-butoxycarbonyl)amino)-1-fluoro-2-hydroxypropyl)(1.1-diethoxyethyl)phosphinate(Intermediate to the Compound According to the Example 7)

To solution of ethyl(3-(N-(tert-butoxycarbonyl)amino)-1-fluoro-2-oxopropyl)(1,1-diethoxyethyl)phosphinate(0.7 g, 1.8 mmol) in methanol (30 mL) at −5° C. under a nitrogenatmosphere was added sodium borohydride (76 mg, 2.0 mmol) in oneportion. A slight exotherm occurred; however, the internal temperaturewas maintained below −2° C. The reaction mixture was stirred at 0° C.for 1 h. The reaction mixture was quenched with saturated aqueous sodiumhydrogen carbonate (5 mL). The crude mixture was concentrated underreduced pressure. The crude residue was extracted with ethyl acetate (30mL), washed with saturated aqueous sodium chloride solution (5 mL) anddried over anhydrous magnesium sulfate. Removal of solvent under reducedpressure afforded the crude product as a pale yellow oil (580 mg).Purification by column chromatography afforded 2 fractions, whichappeared to be consistent with different diastereomers of ethyl(3-(N-(tert-butoxycarbonyl)amino)-1-fluoro-2-hydroxypropyl)(1,1-diethoxyethyl)phosphinate.The less polar fraction appeared to be a 1:1 mixture of twodiastereomers, (210 mg, 29%). Whereas the more polar fraction waspredominantly one diastereomer as judged by ¹H NMR analysis (190 mg,26%). ¹H NMR of the more polar compound (300 MHz, CDCl₃) δ 5.32–5.04 (brs, 1H), 4.88–4.82 (m, 0.5H), 4.72–4.68 (m, 0.5H), 4.40–4.08 (m, 4H),3.90–3.26 (m, 6H), 1.66–1.52 (m, 3H), 1.50–1.32 (m, 3H), 1.44 (s, 9H),1.30–1.12 (m, 6H).

EXAMPLE I20 Ethyl3-[(diethoxymethyl)(ethoxy)phosphoryl]-2-fluorobutanoate (Intermediateto the Compound According to Example 8)

A mixture of ethyl (diethoxymethyl)phosphinate (21.7.0 g, 110 mmol) and1,1,1,3,3,3-hexamethyldisilazane (23.3 mL, 110 mmol) was heated toreflux for 2 h under an argon atmosphere. The mixture was cooled to roomtemperature and a diastereomeric mixture ethyl 2-fluorobut-2-enoate(14.6 g, 110 mmol) was added. The reagents were heated to 80° C. for oneday and 120° C. for 2 hours under an argon atmosphere. The mixture wascooled to room temperature and another portion of trimethylsilylactivated ethyl (diethoxymethyl)phosphinate was added (this had beenprepared from ethyl (diethoxymethyl)phosphinate (21.7.0 g, 110 mmol) and1,1,1,3,3,3-hexamethyldisilazane (23.3 mL, 110 mmol) in the same way asabove). The mixture was heated to 100° C. for three days and stillanother portion of trimethylsilyl activated ethyl(diethoxymethyl)phosphinate was added. The mixture was heated to 100° C.for three days under an argon atmosphere, cooled to room temperature andthen diluted with ethyl acetate (300 mL). The solution was washed with 1N HCl (2×200 mL) and saturated sodium chloride. The organic layer wasdried over MgSO₄, filtered, and evaporated to give 42.0 g of a yellowoil. The residue was purified by chromatography on a wet-packed silicagel column eluting with methylene chloride and then with 98:2 methylenechloride/methanol. The appropriate fractions were combined andevaporated to give 3.6 g (10%) of ethyl3-[(diethoxymethyl)(ethoxy)phosphoryl]-2-fluorobutanoate as a clear oil.¹H NMR (400 MHz, CDCl₃) δ 4.9–5.6 (m, 1H), 4.7–4.8 (m, 1H), 4.2–4.4 (m,4H), 3.6–4.0 (m, 4H), 2.6–2.9 (m, 1H), 1.2–1.4 (m, 12H).

EXAMPLE I21 Ethyl3-amino-2-fluoro-1-methyl-3-oxopropyl(diethoxymethyl)phosphinate(Intermediate to the Compound According to the Example 8)

To a solution of ethyl3-[(diethoxymethyl)(ethoxy)phosphoryl]-2-fluorobutanoate (1.8 g, 5.5mmol) in ethanol (3 mL) was added concentrated ammonium hydroxide (14.8M, 0.5 mL, 7.4 mmol). The solution was stirred for 24 h at 40° C. andthen evaporated to give 1.6 g (97%) of a diastereomeric mixture of ethyl3-amino-2-fluoro-1-methyl-3-oxopropyl(diethoxymethyl)phosphinate as aclear oil. ¹H NMR (400 MHz, CDCl₃) δ 5.7–6.7 (m, 2H), 4.9–5.5.6 (m, 1H),4.7–4.8 (m, 1H), 4.1–4.4 (m, 2H), 3.8–4.0 (m, 4H), 2.8–3.0 (m, 1H),1.2–1.4 (m, 12H).

Pharmaceutical Preparations

The compound according to formula I of the present invention can be usedas an active ingredient in a pharmaceutical preparation for oral,rectal, epidural, intravenous, intramuscular, subcutanous, nasaladministration and administration by infusion or for any other suitableroute of administration. Preferably the way of administration is oral orby injection/infusion.

The pharmaceutical preparations contain a compound of the presentinvention in combination with one or more pharmaceutically acceptableingredients. The finished dosage forms are manufactured by knownpharmaceutical processes. Usually the amount of active compounds isbetween 0.1–95% by weight of the preparation, preferably between 0.2–20%by weight in preparations for parenteral use and preferably between1–50% by weight in preparations for oral administration.

In the preparation of pharmaceutical preparations containing a compoundof the present invention in the form of solid dosage units for oraladministration, the compound selected may be mixed with solidpharmaceutically acceptable ingredients (among these for instancedisintegrating agents and lubricating agents). The mixture is thenprocessed into granules, tablets, capsules or sachets.

Dosage units for rectal administration may be prepared in the form ofsuppositories; in the form of a gelatine rectal capsule; in the form ofa ready-made micro enema; or in the form of a dry micro enemaformulation to be reconstituted in a suitable solvent just prior toadministration.

Liquid preparations for oral administration may be prepared in the formof syrups or suspensions, or in the form of a dry mixture to bereconstituted with a suitable solvent prior to use.

Solutions for parenteral administration may be prepared as a solution ofa compound of the invention in a pharmaceutically acceptable solvent andare dispensed into ampoules or vials. They may also be prepared as a drypreparation to by reconstituted with a suitable solvent extemporaneouslybefore use.

The typical daily dose of the active compound will depend on variousfactors such as for example the individal requirement of each patient,the route of administration and the disease. In general, dosages will bein the range of 1 μg to 100 mg per day and kg body weight, preferably 10μg to 20 mg per day and kg body weight.

Biological Studies

[³H]GABA Radioligand Binding Assay

Rat synaptic membranes were prepared from the whole brain of SpragueDawley male rats essentially as described previously (Zukin, et al.(1974) Proc. Natl. Acad. USA 71, 4802–4807). The [³H]GABA competitionassay, modified from Olpe et al ((1990) Eur. J. Pharmacol. 187, 27–38),was performed in 200 μl TCI (Tris Calcium Isoguvacine) buffer (50 mMTris (tri(hydroxymethyl)aminomethane), pH 7.4, 2.5 mM CaCl₂ and 40 μMisoguvacine) containing 20 nM [³H]GABA (specific activity: 3 TeraBecquerel (TBq)/mmol), test compound or solvent and 80 μg synapticmembrane protein using 96-well plates. After incubation for 12–20 min atroom temperature, incubations were terminated by rapid filtrationthrough a glass fiber filter (Printed filtermat B filters, Wallac),which had been pretreated with 0.3% polyethyleneimine, using a 96-wellplate cell harvester (Skatron or Tomtec). The filters were washed withbuffer containing 50 mM Tris (tris(hydroxymethyl)aminomethane) and 2.5mM CaCl₂, pH 7.4, at 4° C. and then dried at 55° C. MeltiLex B/HSscintillator sheet (Wallac) was melted onto the filter, andradioactivity was determined in a Microbeta scintillation counter(Wallac).

Results and Discussion

The compounds of the present invention were found to have highaffinities and potencies for the GABA_(B) receptor as revealed by lowIC₅₀ and EC₅₀ in the binding and ileum assays, respectively. Thecompounds have also been found to reduce TLOSR when administered i.v. aswell as p.o. in animal models. Contrary to what has been claimed in theliterature for 3-aminopropylphosphinic acid derivatives having a P—Hbond, we found that the compounds of the present invention have highmetabolic stability in animal models. Moreover, CNS side-effects (asmeasured by reduction in body temperature in the mouse) were notobservable or only seen at very high doses. Therefore, the differencebetween therapeutic dose (inhibition of TLOSR in the dog model) and dosecausing side-effects (in the mouse model) was unexpectedly high.

1. A compound selected from the group consisting of:ethyl-3-[(diethoxymethyl)(ethoxy)phosphoryl]-2-fluoropropanoate,ethyl-(3-amino-2-fluoro-3-oxopropyl)(diethoxymethyl)phosphinate,ethyl-(2R)-(3-chloro-2-hydroxypropyl)(1,1-diethoxyethyl)phosphinate,ethyl-(2S)-(3-amino-2-hydroxypropyl)(1,1-diethoxyethyl)phosphinate,ethyl-(2S)-(3-chloro-2-hydroxypropyl) (1,1-diethoxyethyl)phosphinate,ethyl-(2R)-(3-amino-2-hydroxypropyl) (1,1-diethoxyethyl)phosphinate,ethyl-[3-[N-(tert-butoxycarbonyl)amino]-2-oxopropyl](1,1-diethoxyethyl)phosphinate,(2R)-3-(dibenzylamino)-2-fluoro-1-propanol,(2R)3-amino-2-fluoro-1-propanol,tert-butyl-(2R)-2-fluoro-3-hydroxypropylcarbamate,tert-butyl-(2R)-2fluoro-3-iodopropylcarbamate,methyl-(2S)-3-(dibenzylamino)-2-fluoropropanoate,(2S)-3-(dibenzylamino)-2-fluoro1-propanol,(2S)-3-amino-2-fluoro-1-propanol,tert-butyl-(2S)-2-fluoro-3-hydroxypropylcarbamat,tert-butyl-(2S)-2-fluoro-3-iodopropylcarbamate,ethyl-(fluoromethyl)(1,1-diethoxyethyl)phosphinate,ethyl-(3-(N-(tert-butoxycarbonyl)amino)-1-fluoro-2-oxopropyl)(1,1-diethoxyethyl)phosphinate,ethyl-(3-(N-(tert-butoxycarbonyl)amino)-1-fluoro-2-hydroxypropyl)(1,1-diethoxyethyl)phosphinate,ethyl-3-[(diethoxymethyl)(ethoxy)phosphoryl]-2-fluorobutanoate, andethyl-3-amino-2-fluoro-1-methyl-3-oxopropyl (diethoxymethyl)phosphinate.